Medlyn, Michael

Abstract Introduction Checkpoint blockade revolutionized cancer therapy. However, only a small proportion of cancers respond to checkpoint blockade. B7-H3 is a member of the B7 superfamily, which includes both co-inhibitory and co-stimulatory receptors. B7-H3 is upregulated by many tumors which is generally associated with a worse prognosis. However, the exact mechanism by which B7-H3 suppresses the immune response remains elusive. Methods We transfected human HEK293T cells and assessed the binding to recombinant Siglecs via flow cytometry. We generated monoclonal antibody F7 against human B7-H3 and tested its blocking function using B7-H3 transfected HEK293T cells. We examined the extent of degranulation and NK cell-mediated killing on B7-H3 expressing HEK293T cells in vitro, as well as the CART19 cells mediated killing in relapse model of B7-H3 expressing CD19+ leukemia Nalm6 cells NSG mice. Results We demonstrated that sialylated B7-H3 binds to the inhibitory receptor Siglec-9 on immune cells but not to the related protein Siglec-7. A high affinity monoclonal antibody (mAb) F7 against B7-H3 blocks Siglec-9 binding. Functionally, F7 enhances NK cell killing of B7-H3 expressing target cells in vitro. F7 also dramatically enhances CART19 mediated killing of B7-H3 expressing Nalm6 cells in a relapse model in NSG mice in vivo. Conclusion We have now shown for the first time that B7-H3 binds to the inhibitory receptor Siglec-9 and generated a novel monoclonal antibody F7 that target a unique sequence in B7-H3 as compared to other monoclonal antibodies being examined for clinical efficacy. F7 can promote NK cell killing of B7-H3 expressing target cells in vitro and synergize with CART19 to enhance killing of B7-H3 expressing tumor cells in vivo. As B7-H3 is overexpressed in many human cancers, this work is critical toward understanding how B7-H3 mediates immune evasion through Siglec-9 for enhancing anti-tumor immunity. Funding Source R01CA243545-01A1 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

AbstractPatient‐derived cancer organoids (PDOs) hold considerable promise for personalizing therapy selection and improving patient outcomes. However, it is challenging to generate PDOs in sufficient numbers to test therapies in standard culture platforms. This challenge is particularly acute for pancreatic ductal adenocarcinoma (PDAC) where most patients are diagnosed at an advanced stage with non‐resectable tumors and where patient tissue is in the form of needle biopsies. Here the development and characterization of microfluidic devices for testing therapies using a limited amount of tissue or PDOs available from PDAC biopsies is described. It is demonstrated that microfluidic PDOs are phenotypically and genotypically similar to the gold‐standard Matrigel organoids with the advantages of 1) spheroid uniformity, 2) minimal cell number requirement, and 3) not relying on Matrigel. The utility of microfluidic PDOs is proven by testing PDO responses to several chemotherapies, including an inhibitor of glycogen synthase kinase (GSKI). In addition, microfluidic organoid cultures are used to test effectiveness of immunotherapy comprised of NK cells in combination with a novel biologic. In summary, our microfluidic device offers considerable benefits for personalizing oncology based on cancer biopsies and may, in the future, be developed into a companion diagnostic for chemotherapy or immunotherapy treatments.

Abstract The recycling of membrane proteins from endosomes to the cell surface is vital for cell signaling and survival. Retriever, a trimeric complex of VPS35L, VPS26C and VPS29, together with the CCC complex comprising CCDC22, CCDC93, and COMMD proteins, plays a crucial role in this process. The precise mechanisms underlying Retriever assembly and its interaction with CCC have remained elusive. Here, we present the first high-resolution structure of Retriever determined using cryogenic electron microscopy. The structure reveals a unique assembly mechanism, distinguishing it from its remotely related paralog, Retromer. By combining AlphaFold predictions and biochemical, cellular, and proteomic analyses, we further elucidate the structural organization of the entire Retriever-CCC complex and uncover how cancer-associated mutations disrupt complex formation and impair membrane protein homeostasis. These findings provide a fundamental framework for understanding the biological and pathological implications associated with Retriever-CCC-mediated endosomal recycling.